Methods and gel compositions for encapsulating living cells and organic molecules

ABSTRACT

A method for encapsulating biologics within a hydrogel by using an aqueous solution of an isocyanate-functional hydrogel prepolymer which is mixed with an amount of biologics and an aqueous solution containing a dithiol crosslinking agent under physiological pH conditions. An additional bidentate crosslinking agent may be included. The product of such method may be a bioreactor or an assay device having a plurality or different biologics encapsulated at predetermined locations in a substrate.

[0001] This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/281,268, filed Apr. 3, 2001, the disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to systems and methods for formingpolyurethane hydrogels useful for encapsulating biologics, such asliving cells, proteins, enzymes, antibodies and small organic molecules,and to the compositions which result therefrom. More specifically, thepresent invention relates to the formulation and use of a polymerizationprocess employing biocompatible polymers and biocompatiblepolymerization conditions, such as neutral pH, where there ismaintenance of an aqueous environment and preservation ofphysiologically relevant osmolarity throughout the polymerizationprocess, as well as to bioassays utilizing such improved resultantproducts. This invention represents a significant development in the artof encapsulation of certain materials and a significant advancement,from certain standpoints, of the process described in U.S. Pat. No.6,174,683, which is assigned to the assignee of this application.

DESCRIPTION OF PRIOR ART

[0003] The use of enzymes, antibodies, peptides, or other bioactivemolecules, e.g. aptamers, has received increasing attention as tools forscreening in the fields of bioassays and proteomics. As part of thisdevelopment, the use of hydrogel supports for these bioactive materialshas also gained in importance. Hydrogels are defined as water-containingpolymeric matrices. In particular, hydrogels provide a support forbiomaterials that more closely resembles the native, aqueous, cellularenvironment, as opposed to a more denaturing environment that resultswhen proteins or other materials are directly attached to a solidsupport surface using other molecular scale linkages, such as coatings.

[0004] Certain hydrogels have previously been described as matrixsupports for biomolecules and/or living cells, and these includealginates, alginates modified to permit cross-linking, acrylamide-basedhydrogels, and polyethylene oxide-based hydrogels. In general, however,there is frequently difficulty in reconciling the gel polymerization andencapsulation requirements with the gentle conditions requisite formaintaining the viability or activity of live cells or certain activeproteins. In addition, many of the materials suitable for these gentleconditions, e.g. alginate-based polymers, lack the structuralrequirements and/or biostability necessary for broad applications.

[0005] Alginate gels have been widely utilized for immobilization ofeukaryotic cells and proteins. This form of hydrogel is generally benignand biocompatible during the encapsulation process; however, it cansuffer from a lack of structural stability. Alginates are thus sometimescombined with multivalent cations to form more stable, ionicallycross-linked gels. However, upon exposure to physiologically relevantbuffers and environments, divalent cations tend to exchange withmonovalent species, and the polymer often loses structural integrity. Asa result, alginates are somewhat undesirable hydrogels for encapsulatingbiomolecules and living cells. Moreover, the overall manufacturabilityof alginate gels is difficult, further lessening the desirability andapplicability of such a gel system.

[0006] Polyacrylamide hydrogel systems have also received considerableattention as matrices for attaching biomolecules and encapsulationvehicles. For example, Arenkov, et al. (Anal. Biochem. 278, 123-131(2000)), describe gel pad arrays formed by photoinitiated polymerizationof acrylamide/bisacrylamide mixtures using methylene blue as thephotocatalyst. Proteins are then covalently linked to each gel padfollowing application to the micromatrix either by crosslinking withglutaraldehyde or by chemical modification of carbohydrate moietiespresent on select proteins to allow subsequent chemical linkage to thegel support. However, such a method of linkage can be potentially verydamaging to the integrity and/or activity of the protein, and it mayalso require the presence of sugar residues not ubiquitously found onall proteins.

[0007] Alternative to the use of polyacrylamide-based hydrogels are theuse of those composed primarily of polyethylene oxide (PEO)polymerization units. These polymers can offer a number of distinctadvantages in the areas of biocompatibility, diffusion of smallmolecules and manufacturing process control. For example, the graftingof PEO onto serum albumin significantly reduces immunogencity of thenative albumin (Abuchowski, et al. 1977). Hubbell, et al. (U.S. Pat. No.5,573,934 and related patents) teach the use of polyethylene glycolpolymers for encapsulating cells using a dye-based photoinitiated freeradical-based polymerization process.

[0008] In the aforementioned polyacrylamide or PEG polymeric gels,initiation of polymerization requires the addition of a separate,photoactivatable catalyst and/or the addition of free radical-generatingpolymerization accelerators, separate and distinct from the polymercomponents or subunits. Chudzik and Anderson (U.S. Pat. No. 6,156,345)teach the use of polymer initiator groups which are pendant from thepolymerizable groups and thus avoid the separate addition of initiatorcomponents.

[0009] Mixed polymer/alginate systems have also been devised to overcomelimitations inherent in each system alone. For instance, Desai, et al.(U.S. Pat. No. 5,334,640) employ mixtures of an ionically cross-linkedbiocompatible component with a covalently linked component. However, theoverall process remains dependent upon photoinitiated, freeradical-based polymerization.

[0010] Use of methodologies incorporating free radicals as essentialelements within such a process is a generally undesirable feature ofmany of the encapsulation/polymerization techniques in present use. Forexample, in cell encapsulation with acrylamide gels, “polymerization ofacrylamide generates heat and free radicals, causing loss of in thechemiosmotic integrity and enzymatic activity of the immobilized cells”(see Poncelet De Smet, et al. in “Fundamentals of Animal CellEncapsulation and Immobilization”, Mattheus F. A. Goosen, editor, CRCPress, Boca Raton, Fla., 1993, p. 301). It is therefore desirable toprovide a polymerization process which does not use free radicals toinitiate polymerization, thereby avoiding potential harm to encapsulatedcells and biomolecules. It is also desirable to utilize polymers whichhave both structural and mechanical durability in biological situationsand uses, particularly ones which are truly biocompatible, i.e.non-toxic to the encapsulated biomolecule or cell and to the surroundingmedia or host.

[0011] Wood, et al. teach the use of various cross-linking polymersystems, including a polyurethane-based hydrogel formed fromisocyanate-functional prepolymers, to form a cross-linked polymer toencapsulate microbial cells (U.S. Pat. Nos. 4,436,813 and 4,732,851).Also described are methods using polyazetidine prepolymers andcarboxymethylcellulose which can be crosslinked with polyvalent ions.Direct contact of isocyanates with the microbial cells which occurs inthe encapsulation within such a polyurethane-based hydrogel and exposureto other potentially toxic conditions may not be suitable for theencapsulation of certain sensitive biological materials.

[0012] In the '683 patent, a polyurethane-based hydrogel prepolymer isused to simultaneously derivatize biomolecules, such as nucleic acidprobes, within its structure during polymerization. Such apolymerization process can use PEG-based prepolymers and is advantageousfrom its avoidance of free radicals or other agents as a result of itsemploy of water to initiate polymerization. However, because organicsolvents are often employed in the prepolymer formation, derivitizationand/or solubilization, the process may still be toxic to certainsensitive biological materials, such as living mammalian cells.

[0013] In brief, there remains a particular need for truly benign,non-toxic, biocompatible and mechanically robust hydrogel polymers andassociated polymerization methodology in order to encapsulate certainbiologics, such as sensitive proteins, enzymes, antibodies and livingcells, in a useful and economically feasible fashion, which can provideproducts that are well suited for assays and other applications.

SUMMARY OF THE INVENTION

[0014] It is an object of the invention to provide a method forbiocompatible polymerization of isocyanate-modified biocompatiblemacromers to either directly or indirectly encapsulate or coatbiologics, i.e. living cells, proteins, nucleic acids and otherbioactive materials and compounds, including small organic molecules.The polymerization process is truly biocompatible as it employs noorganic solvents. This novel process utilizes thiol-based crosslinkerswhich reduces the crosslinking of biomaterials within the hydrogel,thereby rendering the process capable of encapsulating and attachingsuch biological material in forms particularly suitable for diagnosticand therapeutic use, for example, microarrays of proteins or cells orother organic compounds for high-throughput testing.

[0015] The method of polymerization employs thiol-containingcrosslinkers and selective reaction conditions, specifically neutral pHand aqueous buffers, to preferentially favor the reaction of sulfhydrylgroups, as opposed to amines, as the preferred conjugation nucleophilewhere water is present during polymerization; this provides mild,non-radical reaction conditions that allow gentle encapsulation which isof particular importance to biomolecules and living cells. The porosityof the encapsulating polymer can be advantageously varied, and theencapsulation process permits deposition, onto glass slides or othersurfaces, of discrete hydrogel droplets in spots or layers thatencapsulate cells, proteins or other organic molecules, either directlyor indirectly through binding agents, or alternatively by formingdroplets or spheres that separately encapsulate such biologics.Moreover, the overall encapsulation/polymerization process comprisesfewer steps than comparable methodologies, thereby simplifying andeasing process development. Because the resultant polymers can provideantibody or enzymatic arrays and viable cell encapsulation, thepotential employment of such materials in bioreactors, biosensors,biochips and artificial organs is facilitated. Such encapsulated cellsare expected to serve as a logical extension of bioassay development forcomplex biopathway screening, and encapsulated cells will be usefultools in bioreactors for economically generating complex therapeuticagents and materials. In addition, encapsulated living cells maypotentially serve as artificial organs or biosensors, responding asneeded to altered or toxic environments. Microarrays of encapsulatedcells or other such biologics are also expected to be useful in highthroughput biological testing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a diagrammatic view showing a mechanism of cross-linkingprepolymers.

[0017]FIG. 2 is a diagrammatic view, similar to FIG. 1, of analternative crosslinking reaction embodying various features of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Water is often added to cure or initiate the crosslinking ofisocyanate-functional prepolymers. This is in contrast to processesemploying free radical-based methodology, e.g. UV-inducedphotopolymerization, that is used to generate reactive species suitablefor forming covalent linkages between prepolymer units.Isocyanate-functional groups are covalently linked to a prepolymer ofchoice, and such addition of water produces an active primary amine at acertain frequency by conversion of some isocyanate moieties, based upontemperature and pH. Such primary amines subsequently react with otherisocyanates attached to other prepolymer units, thereby covalentlylinking the prepolymer units together, as illustrated in FIG. 1; this isgenerally representative of certain reactions utilized in the '683patent. This process leads to the generation of an opticallytransparent, urea-based hydrogel, so long as reactivity of theprepolymer and reaction conditions are controlled to prevent gas bubbleformation and/or precipitation of the polymer. During such apolymerization process, various biological entities or small molecules,i.e. biologics, can be present or can be added to create biologicallyactive hydrogels. The term biologics, for purposes of this patentapplication, should be understood to include living cells, proteins,such as antibodies, other bioactive materials, both natural orsynthetic, and small organic molecules which function bioactively. Itcan thus be seen that the size of a biologic may vary substantially and,as explained hereinafter, molecules of small size may desirably beprovided with anchoring moieties.

[0019] The '683 patent describes the addition of primaryamine-derivatized oligonucleotides to isocyanate-functional prepolymersin order to produce oligonucleotide arrays that are attached to a solidsupport surface. An advantageous feature of such a process is that,during the completion of the polymerization reaction between isocyanateprepolymer units, the oligonucleotides will become covalently linked tothe polymer matrix. However, such a method, based upon amineconjugation, may not be suitable for certain sensitive biologics, e.g.certain proteins and living cells. Because primary amines are componentsof all proteins including those present on the surface of living cells,e.g. ligand receptor proteins, ion channel proteins and cell-to-celladhesion proteins, extensive derivatization or conjugation of suchamines directly to the isocyanate-functional prepolymer may lead to theprotein's inactivation, denaturation or altered functionality. It hasnow been found that this possibility is minimized as a result ofemploying a new crosslinking approach that relies primarily upon thiolgroups, instead of amines, for this purpose.

[0020] Thiol-based crosslinking agents serve as mediators of thecross-linking reaction between isocyanate groups on differentprepolymers, as opposed to employing amine functionalities. Of course,in an aqueous environment, a certain percentage of the isocyanate groupswill undergo hydrolysis; however, the primary amines formed as a resultwill have pKa values in the range of 9 to 10. By maintaining a neutralpH, the vast majority of these amines will be protonated and thereforewill not participate in the polymerization process. As a neutral pH, 6.5to 7.5 is preferred, 6.6 to 7.1 is more preferred and approximately pH7.0 is most preferred. The presence of such thiol-containing specieswill cross-link unreacted isocyanate groups so as to effectively carryout the polymerization process.

[0021] One advantageous result of such preferential use of thiolcrosslinkers is the minimization of reactions with the biologics beingencapsulated or immobilized at locations on the molecule whereattachment to the matrix is undesirable. Control of the pH of thepolymerization reaction, which places a restraint upon the nucleophilicreactivity of the amines but not the thiol groups, avoids creation ofextensive links to proteins within the matrix. Proteins are of coursecomposed of a variety of amino acids, some of which contain side chainprimary amines that are potentially reactive during the overallpolymerization process. However, linking to such amine functionalitiesmay well hinder the natural movement and conformation of the proteins,and in the case of living cells, it will likely alter the pattern andresponsiveness of extracellular and plasma membrane proteins. Thepresent method avoids or substantially limits occurrence of such linksand the negative aspects thereof.

[0022] For example, the pKa value for the side chain primary amine ofthe amino acid lysine is quite basic, approximately 10.5, and that forarginine is even more basic, i.e. over 12. If the pH of thepolymerization mixture is maintained approximately neutral, then theproportion of free amine suitable for participating in a nucleophilicaddition, such as that shown in FIG. 1, is less than {fraction(1/1000)}^(th) of the total primary amine population represented bylysine side chains. In contrast, thiol groups remain nucleophilic andvery reactive at neutral pH values. Although cysteine residues inproteins contain a thiol side chain, the frequency of cysteines withinproteins is generally more than 3-fold lower than that of lysine, andwhen present, cysteines are frequently oxidized so as to formintramolecular cystine linkages in native proteins, thereby furtherlowering the number of available sulfhydryl groups. The overall resultis a very substantial reduction in the number of multiple, potentiallydenaturing links between embedded proteins or cells and the polymermatrix; thus, such thiol-mediated crosslinking of hydrogel prepolymersprovides improved formulations for encapsulating sensitive biologicalmolecules and living cells.

[0023] In addition, this encapsulation method, which depends upon thiolreactions, also provides a very effective way of anchoring small organicmolecules, for example organic molecules having a molecular weightbetween 100 and 2000 and particularly those having a molecular weightnot greater than about 500, in a manner so that they fully retain theireffectiveness in the hydrogel. These small molecules are derivatized toplace a thiol group at a location in the molecule where it will notinterfere with the secondary or tertiary configuration of the smallmolecule, for example, at one end of a generally linear molecule.Although the small molecule might be of such a size that it would notnecessarily be retained in an encapsulating matrix of this type, thepresence of the thiol group will result in a linking to an isocyanategroup on the polymer and thus anchor the small organic molecule withinor upon the gel in a manner such that it can assume its normal activeconfiguration. In this manner, the encapsulation method can be used tocreate what might be termed chemical chips, as well as protein chips,cellular chips and the like.

[0024] Isocyanate-functional prepolymers are often prepared fromrelatively high molecular weight polyoxyalkylene diols or polyols thatare reacted with difunctional or polyfunctional isocyanate compounds.Preferred prepolymers are ones made from polyoxyalkylene diols orpolyols that comprise homopolymers of ethylene oxide units or block orrandom copolymers containing mixtures of ethylene oxide units andpropylene oxide or butylene oxide units. In the case of such block orrandom copolymers, at least 75% of the units are preferably ethyleneoxide units. Such polyoxyalkylene diol or polyol molecular weight ispreferably from 2,000 to 30,000 and more preferably from 5,000 to30,000. Suitable prepolymers may be prepared by reacting selectedpolyoxyalkylene diols or polyols with polyisocyanate, at anisocyanate-to-hydroxyl ratio of about 1.2 to about 2.2, so thatessentially all of the hydroxyl groups are capped with polyisocyanate.Aliphatic, rather than aromatic isocyanates, are preferred as theyprovide more easily controlled polymerization. Generally, polyethyleneglycol (PEG), polypropylene glycol (PPG) or copolymers thereof arepreferred. The isocyanate-functional prepolymers being used preferablycontain active isocyanates in an amount of about 0.1 meq/g to about 1meq/g, and more preferably about 0.2 meq/g to about 0.8 meq/g. Shouldrelatively low molecular weight prepolymers, e.g. less than 2,000, beused, they preferably contain a relatively high isocyanate content(about 1 meq/g or even higher). However, the polymerization rate of suchsmaller prepolymers may require more precise control to avoid too rapidpolymerization, and thus would be less preferred for fabricatingmicroarrays and the like. Moreover, prepolymers with a fairly highisocyanate content may have a relatively high content of free aminesafter polymerization, and the positive charges on such aminefunctionalities, at neutral pH, may increase non-specific binding ofnegatively charged biomolecules with the potential of resulting inhigher levels of undesirable background signals. Thus, higher molecularweight prepolymers which contain a relatively low isocyanate content arepreferred.

[0025] In order to enhance the diffusability of large biologicalmolecules, it may be desirable to use low ratios of prepolymer (3-5%)relative to the total volume of the ultimate formulation. Suchrelatively low percentages aid in producing hydrogel compositions havingthe desired porosity for use in assays, bioreactors and the like. Asmentioned above, the viability of entrapped biological molecules isenhanced through minimization of the involvement of amine groups byemploying crosslinkers with thiol functions and maintaining aphysiological pH of about 7.0, where a large percent of amines (pKa=˜10)will be present as protonated species which do not react with theisocyanate functionalities. Although such an arrangement in someinstances could potentially result in incomplete curing of theprepolymer, the nucleophilic activity of thiols towards isocyanates isunaffected at such pH so curing can be completed, and the overall resultis one of a significant advancement in formulating PEG and/or PPG-basedhydrogels for encapsulating biomaterials.

[0026] Short-chain dithiol crosslinkers, such as 1,4-dithiothreitol(mw=154), produce a fairly high speed polymerization that needs to beslowed and carefully controlled to avoid precipitation. Longer dithiolcrosslinkers provide formulations for hydrogel polymerization that aremore easily controlled. Crosslinkers having a back-bone of PEG and/orPPG units are one class of dithiols that provide biocompatibility andstructural advantages, and such crosslinkers of molecular weight betweenabout 500 and 10,000 are preferred, with those between about 2,000 and6,000 being more preferred and those between about 3,000 and 4,000 beingmost preferred. For example, PEG-(thiol)₂ (Shearwater Polymers, Inc.)having a mw=3,400 and thiol groups at the ends of the chains, may beused with Hypol PreMa G-50 (Hampshire Chemical Corp., which has analiphatic isocyanate content of ˜0.35 meq/g), and by varying the ratiobetween two such starting materials, it was found that the speed ofpolymerization can generally be effectively controlled at pH 7.0. Themolar ratio of dithiol crosslinker to isocyanate (from the prepolymer)is preferably not higher than about 0.3 dithiol per isocyanate, and morepreferably not higher than about 0.2 dithiol per isocyanate.

[0027] Formulations having a ratio significantly lower than 0.1 dithiolper isocyanate, e.g. 0.05 or below, might not polymerize promptly and/orcompletely at pH 7.0 without the inclusion of an auxiliary crosslinker.Thus, formulations having a ratio slightly less than about 0.1 dithiolper isocyanate are preferably supplied with an auxiliary bidentatecrosslinker having two different isocyanate-reactive functional groups,one of which is preferably thiol, e.g. cysteine which has a side chainthiol group and a less reactive primary α-amine group which is of coursemore nucleophilic than the α-carboxyl under these conditions. Otherbidentate crosslinkers that might be used include 2-mercaptoethanol,2-aminoethanethiol, homocysteine, 2-mercaptopropanol and other shortchain compounds having a thiol group and another nucleophilic group.Morever, even when an adequate amount of the dithiol crosslinker isprovided, it has been found that the provision of an auxiliary bidentatecrosslinker can be advantageous in controlling the polymerizationreaction in obtaining completion within desirable time limits and inobtaining hydrogel compositions that are stable, have a high watercontent and excellent structural strength. Accordingly, the employmentof the combination of a dithiol crosslinker of relatively high molecularweight, e.g. about 2,000 to 6,000 mw, and a bidentate crosslinker ofmuch lower molecular weight, preferably below about 300 mw, ispreferred. It was found that this addition of a moderating bidentatecrosslinker having two different reactive groups (e.g. cysteine)provides a novel and powerful means by which polymerization can beeffectively controlled, and such is diagrammatically illustrated in FIG.2, which also indicates that crosslinking in this manner eliminates alarge amount of CO₂ that would otherwise be created in normalcrosslinking. When such an auxiliary crosslinker is used, it isgenerally used in a molar amount from about 1 to 3 times the molaramount of the dithiol, and preferably from about 1.5 to about 2.5 timesthe moles of the dithiol crosslinker, in which amount it has been foundto moderate the polymerization reaction and result in satisfactorycuring.

[0028] The inherent reactivity of prepolymers of this general typeallows the use of chemically functional surfaces to also achievecovalent attachment of the polymer to a substrate during polymerization.Such surfaces may be provided upon substrates which will facilitate thehandling and instrumented examination of the polymerized hydrogel andencapsulated biological matter; for example, fabrication of a microarraycontaining different bioactive material encapsulated into individualspots or regions of polymerized hydrogel placed in a known pattern onsuch a substrate.

[0029] Neutral pH is preferably maintained throughout the polymerizationprocess by the use of 50 mM phosphate buffer supplemented with NaCl,typically 10 mM to 80 mM; osmotic pressure is preferably maintained atphysiological levels, approximately 300 milliosmoles. It is found thatsuch formulations can be made without using organic solvents by mixingisocyanate-derivatized prepolymers rapidly in phosphate buffer/NaCl andthen rapidly adding a premixed solution of cells or proteins and dithiolcrosslinker in phosphate buffer/NaCl. The polymerization process willthen generally occur within 20 to 60 minutes, typically less than 30minutes, during which time the cells or proteins remain in a hydratedand an osmotically balanced state at physiological pH. Preferably, pHand osmolality are maintained between 6.9 to 7.6 and between 250 to 400mOsm/kg, respectively. Once cured, polymer sites containing theencapsulated biologics are easily washed, and manipulated.

[0030] Optical examination of these thiol-crosslinked hydrogels revealsoptical clarity with no background fluorescence attributable to the gelformulation and generally similar optical properties to hydrogelformulations described in the '683 patent. However, in the '683processes, it was often very important to carefully control the rate ofCO₂ evolution to avoid some opacity. The present process which usesdithiol crosslinkers in combination with bidentate modifiers inherentlyminimizes CO₂ evolution, as mentioned before with respect to FIG. 2, andcan produce an optically transparent hydrogel with essentially nodifficulty.

[0031] To show that these polyurethane hydrogels are suitable forencapsulating a wide range of biologics, encapsulation of livingeukaryotic cells was first examined. One criteria for the success ofencapsulation of living cells is an assessment of continued cellviability, and typically, trypan blue exclusion is a techniquefrequently favored by biologists to easily determine cell viability.However, because the hydrogel absorbs a significant amount of the trypanblue dye, determination of cell color and intensity of the intracellulardye using this method was unreliable. As an alternative, AlamarBlue™(Trek Diagnostic Systems, Inc.) was used. AlamarBlue is a dye thatbecomes fluorescent upon reduction by metabolic processes. Goatlymphocytes, having been determined by trypan blue exclusion to haveboth viable and dead cells present within the cell mixture, were chosen,and the prepolymer and cell suspension/dithiol crosslinker/bidentatecrosslinker were mixed. The lymphocytes became encapsulated within thethiol-crosslinked hydrogel when droplets of the mixture were depositedas spots (approximately 300-1,000 microns in diameter, with a heightequal to or greater than 20 microns) upon glass slides and then cured ina high humidity chamber at room temperature; mixing and curing took justless than 20 minutes. Preferably, the relative humidity (RH) is at leastabout 90% and more preferably is about 95% or higher. With the spotsfirmly attached to the glass slide, the slide was incubated for threehours at 37° with RPM 1640 media followed by incubation for 1.5 hourswith AlamarBlue™ dye that had been mixed one part to twenty with RPM1640media. After leaving the slide for 30 minutes in the dark, visualizationof the spots using an epifluorescence microscope revealed brightlystained individual cells against a moderately fluorescent hydrogelbackground. Visible light revealed cells within the gel which were notbrightly stained, which were presumably the aforementioned dead cellsalready present within the cell suspension. Hydrogel-only spots treatedin an analogous fashion had no visible fluorescence. Therefore,AlamarBlue™ is felt to be a useful tool for accessing cell viabilitywithin these polyurethane-PEG hydrogels, and the hydrogel itself and thepolymerization process were shown to be biocompatible by the maintenanceof viable cells.

[0032] Encapsulation of proteins was also examined, formulating the gelessentially as just described. Protein encapsulation was demonstrated bythe sequestration/encapsulation of anti-transferrin antibody within thegel matrix during polymerization. Verification of the antibody'sfunctionality was demonstrated by the specific binding of fluorescentdye-labeled transferrin to those sites containing the anti-transferrinantibody and not at other sites containing different antibodies or noantibodies.

[0033] As additional embodiments, encapsulated living cells and/orproteins within such a thiol-crosslinked hydrogel might be deposited asspots or regions upon support surfaces, such as glass slides, or withinmicrowells or microchambers, such as would be present in standard 96well, 384 well or 1536 well microtiter plates. Distinct advantages arepresent with both approaches. In depositing a number of discrete spotsupon a single surface, each spot might contain a different entity,allowing a single incubation followed by the supply of wash solutions tocontact all spots simultaneously. The use of individual microchamberswould allow robotic handling of the plates and permit the use of lowvolumes of individual test solutions at each well. Combinations of thesetwo approaches may also be used whereby individual chambers, arranged ina standard 96 well array or similar format, are each supplied with oneor more hydrogel spots containing different entities.

[0034] Devices employing such arrays might be employed as combinatorialchemical or drug screening devices, antibody arrays, peptide arrays,cell arrays, enzymatic activity arrays, or DNA or other polynucleotidearrays that will be selective for binding to related proteins or otherbiomolecules. In addition, encapsulated cells or biomolecules coatedonto the walls of microcapillary tubes will function as flow-throughdevices having single or multiple channels, which might be employed asscreening devices or as biosensors on systems, such as in liquidchromatography or in “lab-on-a-chip” devices. Signal readout from suchdevices might be via binding of fluorescent proteins or of antigens, tobe measured by subsequent antibody-based detection methods (possiblyemploying additional arrays), or via reaction with endogenousbiopathways which will result in the formation of a detectable species,e.g. enzymatic conversion of a substrate to a fluorescent dye molecule,or change in the electrical properties, e.g. conductivity, of the celland/or surrounding matrix resulting from exposure to the specific agent.In particular, the addition of either a redox agent to the gel or theaddition of an electrically conductive polymer may enable signaldetection by electrical, non-photonic, means.

[0035] The working examples which follow include the best mode presentlyknown for providing formulations and encapsulation methods embodyingparticular features of the invention; however, they should be understoodnot to constitute limitations upon the scope of the invention which isof course defined by the claims that are set forth hereinafter.

EXAMPLE 1

[0036] Solution A was prepared by mixing 0.075 g of Hypol PreMa G-50(Hampshire Chemical Corp.) and 1.5 mL of 50 mM aqueous phosphate buffer,at pH 7.0 with 80 mM sodium chloride. Solution B was prepared bydissolving 30 mg of PEG-(thiol)₂ (mw=3,400) and 2 mg of free basecysteine (Sigma Chemical Co.) (mw=121) in 1 mL of 50 mM phosphatebuffer, at pH 7.0 with 60 mM sodium chloride. Solution C was prepared bymixing 100 μL of Solution B with 10 μL of goat lymphocytes in Dulbecco'sphosphate-buffered saline. Finally, 200 μL of Solution A was mixed with50 μL of Solution C, and the resulting solution was microspotted ontoamine-treated glass (Silanated Slides, Cell Associates, Inc.) with theuse of 5 microliter glass microcapillary tubes. The hydrogel spots werepolymerized carefully in a humidity box, at about 95% RH, to avoiddehydration. This formulation polymerized within 5-10 minutes. Afterpolymerization, the hydrogel spots were physically stable and stronglyattached to the glass slide; they were immediately treated withDulbecco's modified phosphate-buffered saline solution and incubated ateither room temperature or at 37° for about 3 hours in RPM1640 cellmedia. The viability of lymphocytes was examined by means of theAlamarBlue staining method described previously. They were incubated for1.5 hours with RPM1640 media plus the dye and then examined with a lightmicroscope; viable, encapsulated cells were observed.

EXAMPLE 2

[0037] Solution A was prepared by mixing 0.1 g of Hypol PreMa G-50(Hampshire Chemical Corp.) and 1 mL of 50 mM phosphate buffer, at pH 7.0with 80 mM sodium chloride. Solution B was prepared by the sameprocedure as in Example 1. Solution C was prepared by mixing 40 μL ofSolution B with 70 μL of goat lymphocytes in Dulbecco'sphosphate-buffered saline. Finally, 100 μL of Solution A was mixed with100 μL of Solution C, and the resulting solution was microspotted usingthe same procedure as in Example 1. The formulation polymerized in 5minutes, and the hydrogel spots were treated with Dulbecco's modifiedphosphate-buffered saline solution and incubated at 37° C. for 1 day to3 days in RPM1640 cell media. The viability of lymphocytes was examinedwith a light microscope using AlamarBlue which demonstrated viableencapsulated cells.

EXAMPLE 3

[0038] Solution A was prepared by mixing 0.1 g of Hypol PreMa G-50(Hampshire Chemical Corp.) and 1 mL of 50 mM phosphate buffer, at pH 7.0with 80 mM sodium chloride. Solution B was prepared by the sameprocedure as in Example 1. Solution C was prepared by mixing 40 μL ofSolution B with 70 μL of E. coli in Dulbecco's phosphate-bufferedsaline. Finally, 100 μL of Solution A was mixed with 100 μL of SolutionC, and the resulting solution was placed into a disposable culture tube.The formulation polymerized in 5 minutes, and the hydrogel was treatedwith Dulbecco's modified phosphate-buffered saline solution andincubated at 37° C. for 1 day in RPM1640 cell media. Viability andgrowth of E. coli were confirmed by observing turbidity in the hydrogelafter 1 day.

EXAMPLE 4

[0039] Solution A was prepared by mixing 0.1 g of Hypol PreMa G-50(Hampshire Chemical Corp.) and 1 mL of 50 mM phosphate buffer, at pH 7.0with 80 mM sodium chloride. Solution B was prepared by dissolving 30 mgof PEG-(thiol)₂ (mw=3,400) and 2 mg of free base cysteine in 1 mL of 50mM phosphate buffer, at pH 7.0 with 60 mM sodium chloride. 25 μL ofSolution A, 10 μL of Solution B, 5 μL of 50% trehalose in DI water and10 μL of anti-transferrin antibody (goat anti-human transferrin, 5mg/ml, protein-G purified)(Calbiochem) were mixed, and the resultingsolution was microspotted onto an amine-treated glass slide so as toform spots 300 μm to 1,000 μm in diameter and at least 20 μm in height.Other similar spots were created without the addition of theanti-transferrin antibody. The hydrogel spots were carefully polymerizedin a humidity box at room temperature and 95% RH, and afterpolymerization, the hydrogel spots were found to be physically stableand well attached to the glass slide. The slide was treated with a PBSbuffer containing 1% Bovine Serum Albumin (Sigma Chemical Co.) and 0.1%Triton X-100 (Boehringer Mannheim) for 1 hour at room temperature.Anti-transferrin in the hydrogel was interacted with Cy3-labeledtransferrin (1 μg/ml in 1% bovine serum albumin, 0.1% triton X-100 inphosphate buffered saline) for 1 hour and then visualized; itdemonstrated that there was specific binding of fluorescent dye-labeledtransferrin at sites containing the anti-transferrin antibody and not atother sites containing different antibodies or no antibodies.

EXAMPLE 5

[0040] Solution A was prepared by mixing 0.1 g of Hypol PreMa G-50(Hampshire Chemical Corp.) and 1 mL of 50 mM phosphate buffer at pH 7.0.Solution B without salts was prepared by the same procedure as inExample 1. Solution C was prepared by mixing 40 μL of Solution B with 70μL of 234 μmL-alpha-cysteine-N-[8-(1,2,3,4-tetrahydro-acridin-9-ylamino)-octyl]-amide(mw=428.26), an acetylcholine esterase inhibitor, in 50 mM phosphatebuffer at pH 7.0. Finally, 100 μL of Solution A was mixed with 100 μL ofSolution C, and the resulting solution was microspotted using the sameprocedure as in Example 1. The formulation polymerized in 5 minutes, andthe hydrogel microspots were treated with cy-3 labeled acetylcholineesterase. This testing confirmed presence and functionality ofacetylcholine esterase inhibitor in the hydrogel.

[0041] Although the invention has been described with regard to certainpreferred embodiments, it should be understood that changes andmodifications as would be obvious to those having ordinary skill in thisart may be made without deviating from the scope of the invention whichis set forth in the claims appended hereto. The inclusion of additionalpolymers or modifications to the above-described polymer might permiteither cell proliferation or increased viability of select cell typeswithin the matrix. For example, peptide linkages within such a polymermay be specifically crafted to dissolve upon exposure to extracellularmatrix proteases generated by the encapsulated cell, thereby dissolvingthe polymeric matrix as needed to permit cell expansion and growth.Alternatively, other polymers or agents, such as collagen, might beadded to such a polymeric blend to aid cell viability by use of specificadhesion factors and/or binding methods between encapsulated cells andsurrounding support. In contrast to spotting the hydrogel compositionsonto a solid surface, hydrogel microbeads may be formed whichencapsulate biologics. As one example, after mixing the prepolymer withthe crosslinker and biologics, the polymer/cell (or protein) mix isadded to a non-miscible liquid, such as an oil, while curing isoccurring to cause microbeads of various dimensions to be formed.Separation from the oil or other suspending liquid yields a slurry ofbeads suitable for use in bioreactors, assay devices, artificial organs,biosensors or the like. Moreover, multiple layers of encapsulated cells,proteins or other bioactive molecules might be used to construct complexmaterials having unique overall properties. Alternatively, dyes or otheragents might be added to the encapsulating polymer to facilitatesubsequent identification of the encapsulated cell type if heterogeneousmixtures of cells are to be employed. Such a cell identificationmechanism, combined with a chromaphore-based or fluorescent-basedresponse from specific cells in response to added agents, e.g.expression of green fluorescent protein in response to specific cellsignaling pathway activation by a ligand or drug, permits the screeningof large populations of heterogeneous cells in a rapid and facilefashion.

[0042] The disclosures of all U.S. patents mentioned hereinbefore areincorporated herein by reference. Particular features of the inventionare set forth in the claims which follow.

1. A hydrogel polymerization system for the encapsulation of biologicscomprising: (a) an aqueous solution of polyethylene glycol,polypropylene glycol, or a copolymer thereof, having isocyanateendgroups, (b) an aqueous solution of a thiol-functional crosslinkingagent, and (c) a quantity of biologics for solution or suspension insolution (a) or (b).
 2. The hydrogel polymerization system of claim 1wherein the thiol-functional crosslinking agent comprises athiol-functional crosslinking agent and a bidentate crosslinking agenthaving one thiol group and another different isocyanate-reactive group.3. The hydrogel polymerization system of claim 2 wherein the bidentatecrosslinking agent comprises cysteine.
 4. The hydrogel composition ofclaim 1 wherein said biologics are selected from the group consisting ofproteins, nucleic acids, living cells, and organic molecules having amolecular weight between 100 and
 2000. 5. The hydrogel composition ofclaim 1 wherein said thiol-functional crosslinking agent comprises abackbone of polyethylene glycol, polypropylene glycol, or a copolymerthereof and has a molecular weight between about 2,000 and about 6,000.6. A hydrogel biologic composition comprising: (a) polyethylene glycol,polypropylene glycol, or a copolymer thereof which containsthio-urethane groups that form isocyanate-thiol crosslinks that providea stabilized hydrogel, and (b) a biologic which is bioactiveencapsulated within said stabilized hydrogel.
 7. The hydrogelcomposition of claim 6 wherein said stabilized hydrogel is opticallytransparent.
 8. The hydrogel composition of claim 6 wherein saidbiologic is selected from the group consisting of proteins, nucleicacids, living cells, and organic molecules having a molecular weightbetween 100 and
 2000. 9. The hydrogel composition of claim 6 whereinsaid biologic is an antibody.
 10. A biochip wherein the hydrogelcomposition of claim 6 is formed as a plurality of discrete spotsattached to a substrate.
 11. The biochip of claim 10 wherein said spotsare spatially arranged to form an array.
 12. The biochip of claim 11wherein the plurality of spots include some which contain at least twodifferent biologics at known locations within said array.
 13. A methodof encapsulating biologics within a hydrogel comprising: (a) providingan aqueous solution of an isocyanate-functional hydrogel prepolymer; (b)providing an amount of biologics; (c) providing an aqueous solutioncontaining a thiol crosslinking agent having at least two thiol groups;(d) mixing said solutions and said biologics to initiate polymerizationunder physiological conditions of pH and ionic strength and create apolymerizing hydrogel; and (e) dispensing said polymerizing hydrogelinto a desired physical form.
 14. The method of claim 13 wherein saidamount of biologics is mixed with the solution of thiol crosslinkingagent prior to mixing with said prepolymer solution.
 15. The method ofclaim 13 wherein the thiol crosslinking agent comprises a backbone ofpolyethylene glycol, polypropylene glycol, or a copolymer thereof andhas a molecular weight between about 2,000 and about 6,000.
 16. Themethod of claim 13 wherein the crosslinking agent solution additionallycomprises a bidentate crosslinking agent having one thiol group andanother different isocyanate-reactive group.
 17. The method of claim 16wherein the bidentate crosslinking agent comprises cysteine orhomocysteine.
 18. The method of claim 13 wherein the polymerizinghydrogel is dispensed onto a solid substrate so as to result inattachment thereto of the hydrogel-encapsulated biologics.
 19. Themethod of claim 13 wherein the polymerizing hydrogel is repeatedlydispensed onto multiple, pre-identified areas of a substrate to formdiscrete spots arranged as an array.
 20. The method of claim 19 whereindifferent of said spots at known locations contain at least twodifferent biologics.
 21. The method of claim 13 wherein the biologicscontain thiol-reactive groups for immobilization thereof to thepolymerizing hydrogel.
 22. A bioassay comprising the steps of: (a)providing a biochip which comprises a substrate having a hydrogelcomposition bound thereto in at least two different regions, each regionhaving a thickness of at least 20 micrometers and comprising polymersselected from the group consisting of polyethylene glycol, polypropyleneglycol and copolymers thereof, characterized in that said hydrogelcomposition contains thio-urethane groups formed as a part of crosslinksbetween said polymers as a result of reaction with crosslinkers thatundergo isocyanate-thiol reactions, wherein said hydrogel compositionfurther includes biologics encapsulated therewithin; (b) contacting thebiochip with an analyte solution, and (c) detecting the interactions ofthe biochip with the analyte solution.
 23. The bioassay of claim 22wherein said hydrogel composition is exposed to a second analytesolution as a part of said detecting step.
 24. The bioassay of claim 22wherein said analyte solution contains a marker which binds to saidbiologic which is a target and wherein the step of detecting comprisesdetecting the marker bound to the target.
 25. The bioassay of claim 24wherein the marker is capable of fluorescence and wherein the step ofdetecting the bound target comprises detecting fluorescence from themarker.
 26. A method of preparing a hydrogel composition having abiologic encapsulated therein, which method comprises the steps of: (a)providing an isocyanate-functional hydrogel prepolymer, a biologic, anda crosslinlker having at least two thiol groups that will react withsaid isocyanate-functional hydrogel prepolymer, (b) mixing saidisocyanate-functional hydrogel prepolymer, said biologic and saidcrosslinker in an aqueous solution, and (c) maintaining said mixture ofstep (b) so as to cause said biologic to be encapsulated within apolymerized hydrogel wherein said biologic remains bioactive.
 27. Themethod according to claim 26 wherein said mixture is maintained at a pHof between about 6.5 and about 7.5.
 28. The method according to claim 26wherein said aqueous solution also contains a water-soluble bidentatecrosslinker having two different isocyanate-reactive nucleophilicgroups.
 29. A hydrogel composition having a biologic encapsulatedtherein, which comprises: an isocyanate-functional hydrogel prepolymer,a biologic, and a crosslinker having at least two thiol groups, saidisocyanate-functional hydrogel prepolymer having been crosslinked in thepresence of said biologic by reaction with said crosslinker in anaqueous solution at physiological conditions and, as a result of saidcrosslinking, said biologic is encapsulated within a stable polymerizedhydrogel in a manner so that said biologic remains bioactive.